Interactions between the retinal pigment epithelium (RPE) and choroidal endothelial cells (CECs) are important in neovascular age-related macular degeneration (AMD). Events that occur prior to the development of choroidal neovascularization (CNV) in AMD include the activation of CECs to migrate toward a chemotactic gradient in the sensory retina and the loss of integrity of the blood retinal barrier created by RPE. Two complementary sets of findings during the previous funding period - (a) that the active GTP-bound form of Rap1, a GTPase of the Ras superfamily, is important in RPE barrier integrity, whereas knockdown of Rap1 isoforms lead to larger CNV in laser-induced models; and (b) that in CECs, active Rac1, a member of the small GTPases of the Rho family, is important in CEC motility and migration and is a common downstream effector of two signaling pathways in human AMD, VEGF/VEGFR2 and CCl11/CCR3 - provide the bases for the following hypothetical framework that will be tested in the next funding period: In health, RPE junctions disassemble and reassemble as part of homeostasis and the RPE cells release VEGF basally. In early AMD, stresses cause RPE to produce more VEGF. This VEGF activates Rac1 in CECs causing the CECs to migrate and contact the RPE. As a result of CEC-RPE contact, Rap1a has reduced associations with cadherin in adherens junctions, with p22phox, and with protein tyrosine phosphatases, and all these contribute to reduce RPE barrier integrity. CECs then migrate into the sensory retina along a VEGF gradient and proliferate into CNV. Specific Aim 1 is to test how Rap1a associates with junctional proteins to increase RPE barrier integrity, reduce CEC motility and stress fiber formation, and reduce CNV. Specific Aim 2 is to test how active Rap1a regulates endogenous generation of reactive oxygen species and junctional protein phosphorylation in RPE to increase RPE barrier integrity. Specific Aim 3 is to determine mechanisms of crosstalk between CCR3 and VEGF in Rac1-mediated signaling and CEC migration, and the effect of CCR3 inhibition on retinal ganglion and neural cell survival. Methods include: physiologically relevant human RPE-CEC co culture and transmigration models to determine signaling pathways that cause CEC transmigration; engineered adenoviral constructs to test mechanisms in vitro; transgenic Rap1 isoform knockout mice; gene therapy techniques using scAAV and promoters specific to RPE; laser-induced models of CNV; and Micron III fluorescein Interactions between the retinal pigment epithelium (RPE) and choroidal endothelial cells (CECs) are important in neovascular age-related macular degeneration (AMD). PUBLIC HEALTH RELEVANCE: Neovascular age-related macular degeneration is a leading cause of blindness worldwide. Current treatments involve methods to inhibit angiogenesis, which can lead to nutritional deprivation and hypoxia to the outer retina and over time have reduced effect. We use novel approaches to understand why blood vessels grow within the sensory retina in neovascular AMD and then test strategies to maintain vessels within appropriate compartments of the eye. With these approaches we strive to find safe, effective methods that may be used instead of or in addition to current strategies to reduce visual acuity loss in neovascular AMD.